Certain molecules contain functional groups which can absorb and then emit photons. These groups are called fluorophores. A fluorophore absorbs photons of a specific wavelength and energy. Each photon absorbed excites one of the fluorophore's electrons into a higher energy state. The excited electron remains in its high-energy state for a few nanoseconds. While in its excited state, the electron dissipates a small amount of energy via interactions with the rest of the molecule or with the surrounding molecules. The electron then returns to its ground state energy, and, in doing so, emits a photon. This process is known as fluorescence. The emitted photon is of lower energy and hence longer wavelength than the incident photon, due to the dissipative loss of energy to the fluorophore's environment. The difference in energy (or wavelength) is called the Stokes shift.
The Stokes shift is fundamental to fluorescence detection because it allows the small numbers of emission photons to be isolated from the large number of incident excitation photons. For dyes with a small Stokes shift, the excitation and emission spectra have significant overlap, resulting in quenching. Quenching occurs when an emitted photon is reabsorbed by an adjacent fluorophore before it returns to the ground state. Furthermore, an excited electron may also lose its energy via dissipative effects such as vibration. As such, the number of emitted photons may not equal the number of incident photons, reducing the overall signal from the sample. The loss of signal may be quantified by the quantum yield, which is the ratio of emitted photons to incident photons. Generally, the same fluorophore can be repeatedly excited and relaxed, and a single fluorophore can generate many thousands of detectable photons. This allows very sensitive fluorescence detection techniques. However, some fluorophores may be destroyed in the excited state, leading to photobleaching which also has important applications.
The great benefit of fluorescent molecules comes when they are conjugated with biomolecules and used as fluorescent labels for biological imaging or assays. Possible conjugations are proteins, nucleotides, enzymes, fatty acids, phospholipids and receptor ligands. Tissues or cells, containing biomolecules conjugated with fluorophores, can be viewed under a fluorescent microscope. Fluorescent gels and blots can be quantified using a fluorescence scanner. Fluorescent cells or particles can be counted using flow cytometry.
BODIPY dyes are widely used fluorescent dyes that combine a dipyrrinato ligand with a BF2 core, which serves to rigidify the fluorophore, leading to high quantum yields. However, the symmetry of the dye structure results in low Stokes shifts. The core structure has a green fluorescence, but substitutions onto the parent molecule allow 7 different colours from green to red. BODIPY dyes can be readily conjugated with a variety of biomolecules. BODIPY systems have recently displaced common fluorophores such as rhodamine and fluorescein, due to the ease of manipulating their electronic properties. However, one of the problems that has not been circumvented with these systems is the small Stokes shift. Several modifications in the structure have been made either in the ligand or to the atoms attached to boron, but better solutions are still being pursued.